Acetamide stereoisomer

ABSTRACT

The compound of formula (I) 
     
       
         
         
             
             
         
       
     
     is a water-stable, long acting β 2 -selective adrenoceptor agonist useful as a bronchodilator in the treatment of bronchoconstriction associated with reversible obstructive airways diseases and the like. Processes for making the compound of formula (I), as well as related intermediates, are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/157,793, filed Jan. 17, 2014, now allowed, which is a continuation ofU.S. patent application Ser. No. 13/934,996, filed Jul. 3, 2013, nowU.S. Pat. No. 8,664,441, which is a continuation of U.S. patentapplication Ser. No. 12/196,520, filed Aug. 22, 2008, now U.S. Pat. No.8,501,994. U.S. patent application Ser. No. 12/196,520 claims priorityto U.S. Provisional Application No. 60/966,438 filed Aug. 28, 2007. Theentire disclosures of each of these applications are hereby incorporatedherein by reference.

FILED OF THE INVENTION

The present invention relates to a novel acetamide stereoisomer, to aprocess for preparing the acetamide stereoisomer, to a pharmaceuticalcomposition comprising the acetamide stereoisomer and to the use of theacetamide stereoisomer in therapy, in particular in the treatment ofbronchoconstriction associated with reversible obstructive airwaysdiseases including but not limited to asthma, cystic fibrosis andchronic obstructive pulmonary disease, including chronic bronchitis andemphysema.

BACKGROUND OF THE INVENTION

Patients suffering from bronchoconstriction associated with reversibleobstructive airways diseases are generally treated using abronchodilator, to relax the bronchial smooth muscle.

Bronchodilators in use today generally fall into two classes, theβ₂-selective adrenoceptor agonists, such as albuterol (salbutamol),salmeterol and formoterol, and the muscarinic receptor antagonists, suchas ipratropium and tiatropium.

β₂-Selective adrenoceptor agonists may cause adverse effects, and thesemay in part be due to activation of the β₁-adrenoceptor. The selectivityof an agonist for the β₂-adrenoceptor receptor is therefore veryimportant, because it limits the dose that can be given and so affectsthe magnitude of bronchodilations and the frequency of dosing.

A long duration of action is important to patients, not only to minimizethe time spent taking the drug, but also to avoid having to take thedrug during inconvenient times, for example at work, school or duringthe night. Some of the more recent β₂-selective adrenoceptor agonists,in particular salmeterol and formoterol, have a long duration of action,typically about 12 hours. Formoterol has a particular advantage that italso has a fast onset of action. However, formoterol is extremelypotent, which makes it very difficult to formulate, especially foradministration using a metered dose inhaler in a manner that results inuniform drug delivery via aerosol dose after dose (i.e., dose contentuniformity). Furthermore, it is unstable in aqueous solution, whichmeans that solutions for administration using a nebuliser have to bekept refrigerated for a majority of their post-manufacture shelf life.

Formoterol is one of a group of α-aminomethylbenzyl alcohol derivativesfor which patent applications were filed during the early nineteenseventies, for example GB 1 415 256. Perhaps because of the difficultiesassociated with formulating the compound, it took a long time to becommercialized. The compound contains two chiral centers, and hence iscapable of existing and being isolated in four stereoisomeric forms. Thecompound was firstly commercialized as a racemic mixture of the active(R,R)- and inactive (S,S)-isomers, in a dry powder formulation, thenmore recently as the active (R,R)-isomer in a nebuliser solution. It isalso known, for example from U.S. Pat. No. 6,303,145, that the (S,R)isomer of formoterol is active. However, like the (R,R)-isomer, thiscompound is unstable at ambient temperature in aqueous solution andhence nebuliser solutions would need to be stored refrigerated.

SUMMARY OF THE INVENTION

Surprisingly, it has now been found that by replacing the methoxy groupin (S,R)-formoterol with a hydroxy group, and the formyl hydrogen atomwith a methyl group, an isomer having a particularly attractivecombination of properties has been obtained.

According to one aspect, therefore, the present invention provides acompound of formula (I)

or a pharmaceutically acceptable salt thereof.

The compound of formula (I) may also be referred to by the chemical nameN-[2-hydroxy-5-[(1S)-1-hydroxy-2-[[(1R)-2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]phenyl]acetamide.

The isomer of formula (I) has been found to possess particularlyadvantageous properties. In particular, it possesses good, but not veryhigh affinity for the β₂-adrenoceptor, high selectivity for the β₂-overthe β₁-adrenoceptor, a long duration of action and good stability inaqueous solution at ambient temperature.

DETAILED DESCRIPTION OF THE INVENTION

It will be appreciated that the compound provided by the presentinvention is an isomer. This isomer may exist and be isolated inenantiomerically pure form, or in admixture with one or more of itsother isomers. The present invention provides the isomer in any mixtureof isomers other than a racemic mixture, which is described in GB1,415,256. In certain embodiments, the isomer is substantially free ofthe (R,R)-enantiomer, which can exhibit a different potency, resultingin significant variations in the potency of admixtures. It may exist asa 1:1 diastereomeric mixture with the (R,S)-isomer, but is mostpreferably enantiomerically pure (i.e. substantially free of all otherisomers). For example, the isomer may comprise at least 50% by weight ofall 3-acetylamino-4-hydroxy-α-phenylethyl)aminomethyl benzyl alcoholpresent, preferably at least 75%, such as at least 90%, at least 95% orat least 99%.

As used herein, the term “pharmaceutically acceptable salt” refers to asalt prepared from a pharmaceutically acceptable, relatively non-toxicacid, including inorganic acids and organic acids. Suitable acidsinclude acetic, benzenesulfonic, benzoic, camphorsulfonic, carbonic,citric, dihydrogenphosphoric, ethenesulfonic, fumaric, galactunoric,gluconic, glucuronic, glutamic, hydrobromic, hydrochloric, hydriodic,isobutyric, isethionic, lactic, maleic, malic, malonic, mandelic,methanesulfonic, monohydrogencarbonic, monohydrogenphosphoric,monohydrogensulfuric, mucic, nitric, pamoic, pantothenic, phosphoric,phthalic, propionic, suberic, succinic, sulfuric, tartaric,toluenesulfonic, including p-toluenesulfonic m-toluenesulfonic ando-toluenesulfonic acids, and the like (see, e.g., Berge et al., J.Pharm. Sci., 66:1-19 (1977); Stahl and Wermuth, Handbook ofPharmaceutical Salts, Wiley VCH, (2002)). Also included are salts ofother relatively non-toxic compounds that possess acidic character,including amino acids, such as arginine and the like, and othercompounds, such as aspirin, ibuprofen, saccharin, and the like. Acidaddition salts can be obtained by contacting the neutral form of suchcompounds with a sufficient amount of the desired acid, either neat orin a suitable inert solvent. As solids, salts can exist in crystallineor amorphous modifications.

The compounds of the present invention may also be prepared indeuterated form, i.e., in which one or more hydrogen atoms, for exampleon the acetyl group, are replaced with deuterium.

It is also contemplated that the acetyl group in the compound of formula(I) may be replaced with a fluoroacetyl group (i.e. a group in whichone, two or three of the acetyl hydrogen atoms is replaced with afluorine atom). Such compounds may be prepared by a process analogous tothat described herein for the preparation of the acetyl compound.

The acetamide isomer and its pharmaceutically acceptable salts can beprepared by a process, which comprises reacting a compound of generalformula (II)

in which P¹ represents a hydrogen atom or a hydroxyl protecting group,with a compound of general formula (III)

in which P² represents a hydrogen atom or a hydroxyl protecting groupand P³ represents a benzylic amine protecting group, to afford acompound of general formula (IV)

or a salt thereof, followed by removing any protecting groups P¹, P² andP³ and, if desired, forming a pharmaceutically acceptable acid additionsalt.

The protecting groups may be any suitable protecting group, for exampleas described in Green et al., “Protective Groups in Organic Chemistry,”(Wiley, 2^(nd) ed. 1991). Examples of hydroxyl protecting groups includearalkyl groups, such as benzyl, and trialkylsilyl groups, such ast-butyl-dimethylsilyl (TBDMS). Examples of a benzylic amine protectinggroup are benzyl groups optionally substituted on the benzene ring byone or more, for example 1, 2 or 3 optional substituents, for exampleselected from halo, (1-4C) alkyl and (1-4C)alkoxy; for exampleunsubstituted benzyl.

The reaction between the compounds of formula (II) and (III) isconveniently performed by melting the two compounds together, forexample by heating in the range of from 110 to 130° C.

Any protecting groups represented by P¹, P² and P³ may be removed usinga conventional procedure. For example, a benzyl group can be removed bycatalytic hydrogenation in the presence of palladium on carbon, and atrialkylsilyl group by treatment with tetrabutylammonium fluoride.

Compounds of formula (II) can be prepared by reacting a compound offormula (V)

in which Z represents a leaving atom or group, such as a bromine atom,with a base, for example an alkali metal carbonate such as potassiumcarbonate.

Compounds of formula (V) can be prepared by stereoselective reduction ofa compound of formula (VI)

using, for example, borane in the presence of a chiral auxiliary, suchas (1S,2R)-1-amino-2-indanol, followed by reduction of the nitro groupto an amino group and acetylation of the resultant amino group.

Compounds of general formula (III) can be prepared by reacting acompound of general formula (VII)

with boron tribromide, to afford a compound of formula (VIII)

The hydroxyl group may then be protected, for example by reaction with atrialkylsilyl halide, such as t-butyldimethylsilyl chloride.

It will be appreciated that the percentage by weight comprised by thecompound of formula (I) of all3-acetylamino-4-hydroxy-α-phenylethyl)aminomethyl benzyl alcohol presentin the final product of the process will depend upon the enantiomericpurity of the starting materials used and any enantiomeric purificationsteps taken, such as chiral liquid chromatography.

The intermediates of general formula (IV) are believed to be novel andare provided as a further aspect of the present invention.

According to another aspect, therefore, the present invention provides apharmaceutical composition, which comprises a compound of formula (I) ora pharmaceutically acceptable salt thereof, as described herein,together with a pharmaceutically acceptable carrier.

The pharmaceutical composition according to the invention may be adaptedfor administration to patients by any convenient route, such as by oral,mucosal (e.g. nasal, sublingual, vaginal, buccal or rectal), parenteralor transdermal administration. It may be in the form of, for example, asolution, suspension, powder, tablet, aerosol formulation, lozenge,suppository, emulsion, hard or soft gelatin capsule or syrup. Thecompound of formula (I) may be dissolved in the carrier, diluted by thecarrier or supported by the carrier. Thus the carrier may be a supportfor the compound of formula (I), such as a capsule, sachet, paper orother pharmaceutical container.

In one embodiment, the pharmaceutical composition is an aqueous solutionadapted for administration using a nebuliser. The aqueous formulationmay be isotonic and buffered at an optimal pH for stability. The aqueousformulation for nebulization could also be a suspension of nanoparticlesor a micronized suspension of free base or an insoluble salt or acyclodextrin adduct.

In another embodiment, the pharmaceutical composition is an aerosolformulation adapted for administration using a metered dose inhaler, theaerosol formulation comprising the acetamide isomer in crystalline formand a propellant or in solution with an appropriate propellant,combination of propellants or combination of propellant(s) and anacceptable co-solvent or other solubilizing agent.

The propellant may be any suitable propellant used in aerosolformulations, for example, a hydrofluoroalkane (HFA), such as1,1,1,2-tetrafluoroethane (HFA134) or 1,1,1,2,3,3,3-heptafluoropropane(HFA227) or a combination of propellants. HFA134 is preferred. Thepropellant may comprise at least 90% by weight of the aerosolformulation, which may also include, inter alia, inert gases to aide inaerosol formation.

The aerosol formulation may further comprise a surfactant. Thesurfactant serves to stabilize and disperse the acetamide isomer in asuspension, and may also serve as a valve lubricant in the metered doseinhaler. It may be any suitable surfactant used in aerosol formulations.Examples of surfactants used in aerosol formulations are described inU.S. Pat. No. 5,225,183, which is hereby incorporated by reference. Apreferred surfactant is oleic acid. The surfactant, when present, maygenerally be present in an amount of from 1:100 to 1:10surfactant:acetamide isomer, preferably about 1:20.

The aerosol formulation may further comprise a co-solvent. A function ofthe co-solvent in the aerosol formulation is to facilitate dissolutionof the surfactant, which may have poor solubility in the propellant. Itmay be any suitable carrier used in aerosol formulations. A co-solventsuch as glycerol or ethanol may be used. A preferred co-solvent isethanol, especially dehydrated ethanol. The content of ethanol mayconveniently be up to 30% by weight of the aerosol formulation, such asfrom 2 to 6%.

Metered dose inhalers typically comprise a canister containing anaerosol formulation, a metering valve, a valve stem and an actuatorwhich accepts the valve stem. In use, a patient depresses the canisterinto the actuator and inhales, causing a dose of the formulation to beadministered and taken into the patient's lungs.

According to a further aspect, therefore, the present invention providesa metered dose inhaler comprising a canister containing an aerosolformulation as described herein, a metering valve and an actuator.

Preferably the interior surface of the canister is coated, for examplewith a protective polymer, or otherwise treated to minimize chemical orphysical interaction between the formulation and the canister. Theinhaler preferably has an aperture with a diameter in the range of from0.2 to 0.60 mm.

In yet another embodiment, the pharmaceutical composition is in the formof a dry powder suitable for inhalation or insufflation. The compositionmay comprise acetamide isomer crystals alone (e.g. having a mass medianaerodynamic diameter of from 1 to 10 microns, preferably from 2 to 7microns), or acetamide isomer blended, co-precipitated, co-crystallizedor spray dried together with a suitable pharmaceutically acceptablecarrier or carriers. Suitable pharmaceutically acceptable carriersinclude, without limitation, solvates of one or more natural orsynthetic carbohydrates, such as a monosaccharides, disaccharides,trisaccharides, oligosaccharides, polysaccharides, polyols, amino acidsand proteins, and/or in the form of their pharmaceutically acceptableesters, acetals, or salts (where such derivatives exist). The carrier ispreferably lactose, more preferably lactose monohydrate. The dry powdercomposition may be presented in unit dosage form in, for example,capsules or cartridges of e.g. gelatin, or blister packs from which thepowder may be administered with the aid of an inhaler or insufflator.The dry powder composition may be presented in multi dose form meteredwith the aid of an inhaler or insufflator, or pre-metered into discretedoses within the device for serial administrations.

Conveniently, dry powder formulations are administered using multidosedry powder inhalers.

The present invention therefore also provides a multidose dry powderinhaler, comprising a dry powder reservoir containing a dry powderaerosol formulation of acetamide isomer as described hereinabove, and ametering chamber.

The compound of formula (I) according to the present invention may beco-administered with one of more other active ingredients, for exampleselected from steroids, such as beclomethasone, triamcinolone,funisolide, mometasone, budesonide or fluticasone, muscarinic receptorantagonists, such as ipratropium, tiatropium, or glycopyrrolate.Accordingly, in one embodiment, the pharmaceutical composition inaccordance with the present invention may further comprise a steroidand/or a muscarinic receptor antagonist and/or a controller agent orbronchodilator with a novel mechanism.

In another embodiment, the pharmaceutical composition in accordance withthe present invention may further comprise anti-inflammatory agents suchas inhibitors of tumor necrosis factor alpha (TNFα), dipeptidylpeptidase IV, and antibodies to pro-inflammatory interleukins such asIL4 and IL13.

In another embodiment, the pharmaceutical composition in accordance withthe present invention may further comprise mucolytic agents such ascromoglycate, acetylcysteine, arginine, or 2-mercaptoethanesulphonate.

According to another aspect, the present invention provides a method oftreating bronchoconstrictive disease, which comprises administering to apatient in need of treatment an effective amount of a compound offormula (I) or a pharmaceutically acceptable salt thereof.

The bronchoconstrictive disease may be, for example, chronic obstructivepulmonary disease (such as emphysema or bronchitis), cystic fibrosis, orasthma.

The patient may be a human or a non-human mammal, such as a dog, cat,horse, cow, sheep or pig. Preferably, the patient is a human.

The amount of compound administered will depend upon many factors, suchas the species, weight and age of the patient, and the severity of thecondition to be treated. For example, a dose administered to a human maycontain from 75 to 5,000 μg of the acetamide isomer (calculated as thefree base). The dose may be administered, for example, once or twice perday.

According to another aspect, the present invention provides a compoundof formula (I) or a pharmaceutically acceptable salt thereof, for use intherapy.

According to yet another aspect, the present invention provides the useof a compound of formula (I) or a pharmaceutically acceptable saltthereof in the manufacture of a medicament for the treatment of chronicobstructive pulmonary disease.

According to a still further aspect, the present invention provides apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier for the treatment of chronic obstructive pulmonarydisease, or for use as a bronchodilator.

Although the foregoing invention has been described in some detail forpurposes of illustration, it will be readily apparent to one skilled inthe art that changes and modifications may be made without departingfrom the scope of the invention described herein.

EXAMPLES

The following Examples illustrate the invention.

THF refers to tetrahydrofuran, EtOAc refers to ethyl acetate and Et₂Orefers to diethyl ether.

Example 1N-[2-hydroxy-5-[(1S)-1-hydroxy-2-[[(1R)-2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]phenyl]acetamideStep A) (1S)-1-(3-nitro-4-benzyloxyphenyl)-2-bromoethan-1-ol

A cold (5° C.) solution of (1S,2R)-1-amino-2-indanol (400 mg, 2.68 mmol)in THF (160 mL) was added dropwise to a cold (0° C.) solution ofborane-diethylaniline complex (7.0 g, 43 mmol) in THF (20 mL). Aftercomplete addition, the resulting solution was stirred at (0° C.) for 30min then 2-bromo-4′-benzyloxy-3′-nitroacetophenone (20.0 g, 57.1 mmol)was added in three portions over a 30 min period. The resulting solutionwas stirred at <5° C. for 1 h, quenched by dropwise addition of acetone(17 mL) then allowed to warm to ambient temperature overnight. Thereaction mixture was concentrated in vacuo to a residue, which wasdissolved in toluene (100 mL) and washed in succession with 10% H₂SO₄(2×45 mL), H₂O (2×45 mL) and sat. brine (1×40 mL). The organic layer wasdried over MgSO₄, clarified then concentrated in vacuo to a volume of˜40 mL. Heptane (45 mL) was slowly added to give a thick slurry. Thesolid was collected on a filter and washed with heptane (2×5 mL). Thismaterial was dissolved in warm toluene (˜50 mL), the solution wasclarified then diluted with heptane (50 mL). The resulting mixture wasstirred for 30 min, the solids were collected, washed with heptane (2×5mL) then dried to constant weight in vacuo to give 18.9 g (94.0%) of thetitle compound.

Step B) (1S)-1-(3-amino-4-benzyloxyphenyl)-2-bromoethan-1-ol

A solution of the product of step A) (18.7 g, 53.1 mmol) in toluene (40mL) and THF (40 mL) was added to a Parr shaker bottle containing Pt₂O(370 mg). This mixture was shaken under H₂ (50 psi, 344.74 kpa) untilthe reaction was complete (18 h). The catalyst was removed byfiltration, and the filtrate was concentrated to an oil. Columnchromatography (1 kg silica gel packed in and eluted with CH₂Cl₂/MeOH,19:1) gave 11.9 g (69.6%) of the title compound.

Step C) (1S)-1-(3-acetamido-4-benzyloxyphenyl)-2-bromoethan-1-ol

A solution of the product in step B (10.0 g, 31.0 mmol) in pyridine (100mL) was stirred at ambient temperature for 10 min. Acetic anhydride(3.16 g, 30.9 mmol) was added, and the reaction mixture was stirred atambient temperature for 30 min then at 40° C. for 4.5 h. The reactionmixture was cooled to ambient temperature then concentrated in vacuo toa residue. This material was partitioned between CH₂Cl₂ (120 mL) and 10%aq HCl (50 mL). The aqueous layer was extracted with CH₂Cl₂ (50 mL). Thecombined organic layers were washed in succession with H₂O (1×100 mL)and brine (1×100 mL), dried over MgSO₄, clarified then concentrated invacuo to a thick slurry. After dilution with hexanes (60 mL), the solidwas collected on a filter, washed with hexanes (2×20 mL) then dried toconstant weight in vacuo to give 9.4 g (83%) of material as a whitesolid.

Step D) (1S)-1-(3-acetamido-4-benzyloxyphenyl)epoxyethane

A solution of the product in step C (2.5 g, 6.9 mmol) in MeOH (15 mL)and THF (15 mL) was treated with K₂CO₃ (1.3 g, 9.4 mmol), and theresulting mixture was stirred at ambient temperature for 2.5 h. Themixture was concentrated in vacuo to a residue, which was partitionedbetween H₂O (50 mL) and EtOAc (50 mL). The aqueous layer was extractedwith EtOAc (50 mL). The combined organic extracts were washed with H₂O(50 mL treated with a pinch of K₂CO₃ to make it basic), dried overNa₂SO₄, clarified then concentrated in vacuo to a residue that was driedto constant weight in vacuo to give 1.84 g (94%). This material was usedin the next reaction step without further purification.

Step E) [(1R)—N-Benzyl-2-(4-hydroxyphenyl)-1-methylethyl]-amine

To a solution of [(1R)—N-Benzyl-2-(4-methoxyphenyl)-1-methylethyl]amine(5.30 g, 20.8 mmol) in CH₂Cl₂ (25 mL) was added a solution of BBr₃ inCH₂Cl₂ (25.0 mL, 1.0M, 25.0 mmol) slowly over 0.5 h. After the addition,the mixture was stirred at ambient temperature for 22 h. Water (125 mL)was added, followed by the addition of 2.5M aq. NaOH (15 mL) to pH 6.The mixture was extracted with EtOAc (4×200 mL), and the organic layerwas dried (Na₂SO₄) and concentrated. The residue (3.9 g) was trituratedwith CH₂Cl₂ (120 mL) and then concentrated to dryness to give the titlecompound (3.8 g, 76%).

Step F)[(1R)—N-Benzyl-2-(4-t-butyldimethylsilyloxyphenyl)-1-methylethyl]amine

A solution of the product of step E) (3.20 g, 13.3 mmol),tert-butyldimethylsilyl chloride (3.59 g, 23.8 mmol), and imidazole(2.86 g, 42.0 mmol) in DMF (30.0 mL) was stirred at ambient temperaturefor 18 h. The mixture was concentrated to dryness, and the residue waspartitioned between EtOAc (200 mL) and sat. aq. NaHCO₃ (200 mL). Theaqueous layer was separated and again extracted with EtOAc (100 mL). Thecombined organic layer was washed with brine (100 mL), dried withNa₂SO₄, filtered and concentrated to give an oil. The oil waschromatographed on silica gel (100 g, eluted with 1:1 EtOAc:hexanes) togive the title compound (4.0 g, 85%) as a tan oil.

Step G)N-[2-Benzyloxy-5-[(1S)-1-hydroxy-2-[N′-benzyl-[(1R)-2-(4-t-butyldimethylsilyloxyphenyl)-1-methylethyl]amino]ethyl]-phenyl]acetamide

A mixture of the products of Step D) (1.75 g, 6.18 mmol) and F) (2.20 g,6.19 mmol) was heated slowly to 110° C. to give a complete solution. Thereaction solution was heated at 120° C. for 20 h. TLC (EtOAc/hexanes,1:1) showed an estimated 5% of starting material remaining. Heating wascontinued at 120° C. for 5 h then the solution was cooled to ambienttemperature and chromatographed over a column of silica gel (200 g)packed in and eluted with hexanes/EtOAc (2:1). Fractions containingpurified material were combined, clarified then concentrated in vacuo toafford the title compound as a yellow oil, 3.4 g (86%).

Step H)N-[2-Benzyloxy-5-[(1S)-1-hydroxy-2-[N′-benzyl-[(1R)-2-(4-t-hydroxyphenyl)-1-methylethyl]amino]ethyl]phenyl]acetamide

To a stirred solution of the product of Step G) (2.20 g, 3.44 mmol) inTHF (22 mL) at 5-10° C., a solution of tetra-n-butylammonium fluoride inTHF (1.0 M, 4.50 mL, 4.50 mmol) was added. The reaction mixture wasstirred at ambient temperature for 1 h (Note 2), and TLC (1:1EtOAc/hexanes) showed complete consumption of 8. The reaction mixturewas diluted with EtOAc (300 mL) and washed with deionized H₂O (4×200mL), then dried (MgSO₄), filtered, and concentrated to give crude 9 (2.3g). The crude material was purified on a column of flash silica gel (82g, 2.7×35 cm), packed in and eluted with 1:1 EtOAc/hexanes. Fractionscontaining the purified product were combined and concentrated to give acolorless, viscous oil. The oil was coevaporated with Et₂O (3×25 mL) togive the title compound (919 mg, 51%) as a white solid.

Step I)N-[2-hydroxy-5-[(1S)-1-hydroxy-2-[[(1R)-2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]phenyl]acetamide

A mixture of the product of step H) (900 mg, 1.72 mmol), palladium oncarbon (500 mg of 10 wt % Pd) and EtOH (45 mL) was shaken under H₂ (50psi, 344.74 kpa) for 22 h. The mixture was clarified then concentratedin vacuo to an oily residue. The residue was co-evaporated with EtOAc(100 mL) and EtOAc/CH₂Cl₂ (1:1, 100 mL) to give a white solid (536 mg).TLC (4:1 CH₂Cl₂/MeOH) of the solid showed some upper Rf impurities. Thesolid was dissolved in CH₂Cl₂ (20 mL)/MeOH (1 mL), then concentrated inthe cold to ˜10 mL. Hexanes (˜20 mL) were added, and the mixture wasagain concentrated in the cold to give a suspension. A mixture of Et₂O(5 mL), EtOAc (5 mL) and hexanes (5 mL) was added, and the mixture wasconcentrated to give a slurry. This slurry was diluted with hexanes (25mL), and the suspension was stirred vigorously for 0.5 h at roomtemperature. The solid was collected and washed with hexanes (25 mL) togive the title compound (318 mg, 54%); m.p. 97-100° C., with previoussoftening (uncorrected). MS m/z: [M+H⁺] 345.1. ¹H NMR spectrumconsistent with the assigned structure.

β₁ and β₂ Radioligand Binding Assays

The affinity of a test compound for adrenergic β₁ and β₂ receptors isinvestigated by evaluating the ability of the compound to displacespecific binding of [¹²⁵I]-cyanopidolol or [³H]-CGP-12177 at humanrecombinant β₁ and β₂ receptors, respectively (expressed in CHO cells).The IC₅₀ is defined as the concentration that inhibits 50% of specificbinding of the radioligand. The K_(i) is calculated from the IC₅₀ andthe known K_(D) of the radioligand (Cheng and Prusoff's equation).

In this test, the compound of Example 1 was found to afford a K_(i)of >20 μM with only 30% inhibition of specific binding at aconcentration of 20 μM for the β₁ receptor and 2.21 μM for the β₂receptor. The β₁/β₂ binding ratio was found to be >9.

By way of comparison, the values found for arformoterol and the (S,R)isomer of formoterol were 0.155 μM (β₁), 0.004 μM (β₂) and 41 (β₁/β₂),and 2.50 μM (β₁), 0.075 μM β₂) and 33 (β₁/β₂), respectively.

Intrinsic Activity Assessment (β₂)

The intrinsic activity of a test compound is assessed by evaluating itsability to increase cAMP production from human recombinant β₂ receptorsexpressed in CHO cells. Data are expressed as % response relative to aprocaterol-induced cAMP increase.

The compound of Example 1 was found to have an intrinsic activity of71%.

By way of comparison, arformoterol and (S,R)-formoterol were found tohave intrinsic activities of 98% and 91% respectively.

β₁ and β₂ Adrenergic Activity (Functional)

Functional agonism at adrenergic β₁ receptors is demonstrated by apositive chronotropic effect in isolated right atria from Dunkin HartleyGuinea pigs. The concentration that gives 50% maximal effect is theEC₅₀.

Functional agonism at adrenergic β₂ receptors is demonstrated byrelaxation of the spontaneous tone of isolated trachea from DunkinHartley Guinea pigs. The concentration that gives 50% maximal effect isthe EC₅₀.

In these tests, an EC₅₀ could not be determined for the compound ofExample 1 for the β₁ functional assay as only a 32% increase in heartrate was seen at a concentration of 30 μM. However, the compound ofExample 1 was found to have an EC₅₀ of 120 nM for the β₂ receptor. Theβ₁/β₂ functional ratio was found to be >250.

By way of comparison, the values found for arformoterol were 3 nM (β₁),0.041 nM (β₂) and 75.

Stability in Aqueous Buffered Solutions

Solution Preparations: For each test compound, the following solutionsare prepared.

-   -   Solution A is prepared from ˜30 mg of the test compound in 150        mL of 0.005 M citrate buffer, pH 5.0 (˜0.2 mg/mL).    -   Solution B is prepared as follows: approximately 30 mL aliquot        of Solution A is transferred to a separated container and the pH        of the solution is adjusted to pH 3.0 with 1 N HCl (˜0.2 mL).    -   Solution C is prepared as follows: approximately 30 mL aliquot        of Solution A is transferred to a separated container and the pH        of the solution is adjusted to pH ˜8.0 with 1 N NaOH (˜0.2 mL).    -   Note: Because the volume of 1 N HCl or 1 N NaOH used for        adjusting pH was negligible, the concentration of test compound        in Solutions A, B and C were the same.

Storage Scheme

-   -   As soon as the above solutions were prepared, aliquots of each        solution were transferred into 11 vials, of which 9 vials are        stored at −20° C., and one each is stored at 30° C. and 40° C.,        respectively.    -   At each interval listed below, two vials are removed from        −20° C. storage, and stored at 30° C. and 40° C., respectively.    -   The corresponding weeks under the storage condition (30° C. or        40° C.) are shown in the table below.

Week of Vial Removal 0 4 8 10 11 12 Weeks Under Storage Condition 12 8 42 1 0 (30° C. or 40° C.)

On Week 12, the last vial stored at −20° C. is removed and warmed up tothe room temperature, which is the Day 0 Solution.

Sample Analysis: On Week 12, all solutions are assayed by an HPLC methodusing the Day 0 Solution at pH 5 as a standard solution. The testcompounds were assayed by HPLC with UV detection (refer to Table 1 formethod conditions).

TABLE 1 HPLC Method Conditions Parameter Method Detail Column Zorbax SBC8 150 × 4.6 mm Mobile Phase A 0.027M phosphate buffer (pH 3.2)/ACN,95/5, v/v Mobile Phase B 0.027M phosphate buffer (pH 3.2)/ACN, 30/70,v/v Column Temp Ambient Sampler Temp 5° C. Injection Volume 5 μL FlowRate 1.0 mL/min Wavelength PDA 200-350 nm Run Time 55 min Gradient TableTime(min) % A % B 0 100 0 10 90 10 25 80 20 35 65 35 47 0 100 50 100 055 100 0

TABLE 2 Stability of Compound of Example 1 in Aqueous Solution (% ofInitial Concentration (0.20113 mg/mL) in pH 5.0 Citrate Buffer)Condition pH 3.0 pH 5.0 ~pH 8 Time(wk) 30° C. 40° C. 30° C. 40° C. 30°C. 40° C. 0 99.46 99.46 100.25 100.25 100.38 100.38 1 100.03 99.89101.22 102.00 99.64 97.44 2 99.84 100.11 101.07 102.73 99.60 93.75 4101.41 99.54 112.52* 102.94 96.62 86.72 8 100.33 99.18 107.12 122.32*26.79* 72.25 12 100.29 97.17 107.75 112.61* 89.93 61.36 *These resultswere out of trend, but the cause was unknown. The high results at pH 5might be due to evaporation at elevated temperature during storage.

Conclusion: At pH 3 or 5, the compound of Example 1 was found to be verystable for at least 12 weeks when stored at 30° C.

What is claimed is:
 1. An aerosol formulation comprising a compound offormula (I)

or a pharmaceutically acceptable salt thereof.
 2. The aerosolformulation according to claim 1 wherein said compound of formula (I) isin crystalline form.
 3. The aerosol formulation according to claim 1wherein said compound of formula (I) is in solution.
 4. The aerosolformulation according to claim 1 further comprising at least onepropellant.
 5. The aerosol formulation according to claim 4 wherein saidat least one propellant is a hydrofluoroalkane (HFA).
 6. The aerosolformulation according to claim 5 wherein said at least one propellant isselected from 1,1,1,2-tetrafluoroethane (HFA134) and1,1,1,2,3,3,3-heptafluoropropane (HFA227).
 7. The aerosol formulationaccording to claim 4 wherein said at least one propellant comprises atleast 90% by weight of the aerosol formulation.
 8. The aerosolformulation according to claim 1, which further comprises a surfactant.9. The aerosol formulation according to claim 8, wherein said surfactantis present in an amount of from 1:100 to 1:10 surfactant:acetamideisomer.
 10. The aerosol formulation according to claim 1, which furthercomprises a co-solvent.
 11. The aerosol formulation according to claim10, wherein said co-solvent is dehydrated ethanol.
 12. The aerosolformulation according to claim 1, wherein said compound of formula I ispresent in a mixture of isomers ofN-[2-hydroxy-5-[1-hydroxy-2-[[(2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]-phenyl]acetamide,wherein the mixture comprises at least 90% by weight of the isomer offormula I.
 13. The aerosol formulation according to claim 1, whereinsaid aerosol formulation is adapted for administration using a metereddose inhaler.
 14. The aerosol formulation according to claim 1, whichfurther comprises a steroid.
 15. The aerosol formulation according toclaim 1, which further comprises a muscarinic receptor antagonist. 16.The aerosol formulation according to claim 1, which further comprises ananticholinergic.
 17. The aerosol formulation according to claim 1, whichfurther comprises a mucolytic.
 18. The aerosol formulation according toclaim 1, which further comprises an anti-inflammatory agent.
 19. Ametered dose inhaler comprising a canister containing a metering valve,an actuator, and an aerosol formulation comprising a compound of formula(I)

or a pharmaceutically acceptable salt thereof.
 20. A method of treatingbronchoconstrictive disease comprising administering an aerosolformulation comprising a compound of formula (I)

or a pharmaceutically acceptable salt thereof, to a patient in needthereof, wherein said aerosol formulation is administered using ametered dose inhaler.